Although cell culture is often used to study cancer cells and stem cells in vitro, behaviors of these cells may be quite different in culture dishes versus in vivo under their native environment. Lineage tracing is a genetic approach often used in animal models to determine behaviors of cancer cells or tissue stem cells under physiological settings, but its use for human cells is limited. The purpose of this project is to develop a novel lineage-tracing system to enable one-step genetic marking of human (cancer) cells (e.g., in xenograft mouse model), to enable labeling of a pre-defined subset of cells that can be followed in vivo for their clonal behavior, to not introduce mutations (due to genetic marking) that may affect clonal behavior, and to have the capacity to map in vivo fates of multiple stem cell/cancer cell populations in the same tissue/tumor simultaneously. Since the CRISPR/Cas9 genome-editing system can efficiently write insertion or deletion (indel) mutations in pre-designated regions of the human or mouse genome, it is hypothesized that such random indel mutations themselves can serve as stable barcodes for lineage-tracing purpose. Cas9 expression can be controlled in a cell type-specific manner to achieve cell-labeling specificity. An adenoviral system that has been validated by our group for pulse/chase lineage-tracing studies of tissue stem cells can be used to deliver the CRISPR/Cas9 system in vivo. Two Specific Aims are proposed to address this hypothesis: 1) Map contributions of multiple stem cell populations within the same tissue to a specific cell lineage in vivo by multiplexed lineage tracing: Since upon introduction of indel mutations (i.e., barcodes) by CRISPR/Cas9 to different cell types or to different genomic regions, such barcodes can be decoded by next-generation sequencing in a parallel fashion, an important feature of the CRISPR/Cas9-based system may be its capacity for multiplexed lineage tracing. To test this idea, indel mutations will be introduced to the safe-harbor Rosa26 locus (thus mutations introduced are neutral) in mouse mammary epithelial cells in vivo, to determine whether the mammary luminal lineage is maintained by unipotent luminal stem cells, or bipotent basal stem cells, or both, under the physiological setting. 2) Map the in vivo fate of human breast cancer stem cells (CSCs) in a xenograft model: here the feasibility of CRISPR/Cas9-based lineage tracing in human cells will be determined by introducing indel mutations to the AAVS1 safe harbor locus in the human genome. Adenovirus will be used to deliver CRISPR/Cas9 to a CSC population (defined based on a synthetic promoter composed of repeats of SOX2/OCT4-response element) in the human breast cancer cell line, MDA-MB-231, for barcoding. Clonal dynamics of CSCs versus the bulk of cancer cells barcoded by Cas9 will be compared in a xenograft model with or without chemotherapy, or upon serial transplantation. If success, this novel lineage-tracing approach would be transformative for enhancing our understanding of how cancer cells evolve from their cells of origin in vivo and how they become resistant to therapy.